Method for preparing cabbage type rape BnPABP3 promoter and application thereof

A Brassica napus and promoter technology, applied in the field of plant genetic engineering and biology, can solve the problems of harmful ecological environment, unsuitable production practice, unfavorable normal growth of plants, etc.

Inactive Publication Date: 2011-10-19
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of inducible promoters also has certain limitations. The external condition treatment of recipient plants, such as heat shock, hormone treatment, etc., may cause a series of physiological and biochemical reactions in the organism, which is not conducive to t

Method used

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  • Method for preparing cabbage type rape BnPABP3 promoter and application thereof
  • Method for preparing cabbage type rape BnPABP3 promoter and application thereof
  • Method for preparing cabbage type rape BnPABP3 promoter and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Rapeseed P BnPABP3 Expression pattern analysis of driven endogenous genes:

[0057] The rapeseed used in the present invention is Brassica napus L. . The test materials were sown in the field and managed normally.

[0058] Take roots, stems, leaves, flower buds, and siliques from fertile plants with the same growth conditions in the field. Take at least three replicates of each material, and at least one plant for each replicate. After sampling, wrap them in tin foil and place them in liquid nitrogen immediately. medium, stored at -80°C. RNA was extracted (method described above). Fluorescence quantitative PCR instrument for RT TM -Cycler (Boao Biological Co., Ltd.), using rapeseed Actin (accession number AF111812) as an internal standard gene, Actin gene primers are forward primer 5'-CTGGAATTGCTGACCGTATGAG-3' and reverse primer 5'-ATCTGTTGGAAAGTGCTGAGGG-3'. Rapeseed P BnPABP3 The driven endogenous gene primers were 5'-GGGAAGATGATAGGAAGGAAA-3' and 5'-CTGGTGCTCTAAT...

Embodiment 2

[0063] A brassica napus promoter P BnPABP3 The preparation method of promoter, its step is:

[0064] A. Rapeseed P BnPABP3 Promoter primer design:

[0065] Utilize the SDS cracking method to extract the rapeseed DNA (J. Sambrook.D.W.Russell, Huang Peitang et al. Translation Molecular Cloning Test Guide (Third Edition) Science Press), according to the genome walking kit (purchased from Clontech company, product Codenamed Cat.No.638904) operating procedures, genome walking, walking for two rounds of PCR amplification, cloned to obtain rapeseed P BnPABP3 (942bp). The primers used for walking are listed in Table 2.

[0066] Table 2 Genome walking clone P BnPABP3 Primers used

[0067]

[0068] B. Brassica napus P BnPABP3 The promoter preparation is:

[0069] According to the instructions of the genome walking kit (purchased from Clontech, product code Cat. For genomic DNA, four "libraries" of restriction-enzyme-treated DNA ligated with specific adapters were established...

Embodiment 3

[0073] pBI-P BnPABP3 Arabidopsis transformation and PCR detection:

[0074] Arabidopsis thaliana was transformed according to the transformation method in the above literature (Zhang X R. et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature, 2006, 1:1-6). According to the unique kanamycin resistance of the transgenic plants, the green shoots obtained were preliminarily considered to be positive shoots when they were grown on MS (described above) medium containing 50 mg / L kanamycin. After the green seedling grows two true leaves, it is transplanted into vermiculite, and after the inflorescence appears on the plant, a true leaf is taken to extract genomic DNA (described above) by SDS method for PCR identification. The primer sequences are 5′-GAATTGTGAGCGGATAAC-3′ and 5′-ACATAAGGGACTGACCAC-3′; the PCR reaction system is as follows: Genomic DNA template 1μL (about 50ng), 10×Taq enzyme reaction buffer 2ul, 25mMMgCL 2 1.2ul, 2mM...

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Abstract

The invention discloses a method for preparing a cabbage type rape BnPABP3 promoter (PBnPABP3) and the application thereof. The method comprises the following steps of: cloning PBnPABP3 sequence by utilizing the genome walking method, extracting genome DNA by utilizing the SDS cracking method, carrying out enzyme digestion on DNA by respectively using endonuclease DraI, EcoRV, PvuII and StuI in different systems, carrying out the first round of PCR amplification by adopting the product as a template after joint segments are connected with the product, and carrying out the second round of PCR amplification by adopting the 50 times diluent of PCR products obtained in the first round as a template so as to obtain the PBnPABP3. The PBnPABP3 is applied to anther and pollen grains of plant. The promoter has the function of driving exogenous gene expression, the expression parts of the promoter are in the anther and pollen grains of plant, and the promoter has the application potentiality in the aspects of improving the safety of rape edible oil as well as the quality of crops, artificially creating sterile line and restorer line, enriching germ plasm resources, and the like.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology. Specifically related to a Brassica napus BnPABP3 promoter (named P BnPABP3 , the same below), and also relates to a preparation method of Brassica napus BnPABP3 promoter. The present invention also relates to the carrier containing the promoter or its homologous nucleotide sequence and the application of the promoter in genetic engineering of rapeseed and other plants. Background technique [0002] Plant promoters play a key role in the regulation of gene expression. The regulation of gene expression is the result of the comprehensive action of many factors. Generally, according to the sequence of events, gene regulation is divided into transcription level regulation, translation level regulation, and protein processing level regulation. The proteins and RNAs encoded by genes and their secondary metabolites are extremely important for maintaining the whole life acti...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/84A01H5/00
Inventor 刘胜毅石磊董彩华黄军艳刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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